Many complex diseases such as cancer, type 2 diabetes, obesity and autism are on the rise in the United States. They have in common changes in the way ions or small molecules such as nutrients or waste products are moved into or out of the cell. Integral membrane proteins of the of the cation/proton antiporter (CPA) and of the bile/acid sodium symporters BASS) families are responsible for the transport process but the detailed mechanism by which they accomplish their vital function is not understood. Although the three-dimensional structure of some of these transporters are known, key steps in the transport cycle have remained elusive, namely where transported molecules and protons bind and how binding triggers the transport event. Lack of such detailed insights hampers the development of drugs to intervene in diseases. Even more, CPA and BASS transporters have similar structures but perform transport differently. In order to better understand general protein structure/function relationships we need to determine the structural features responsible for the different function. The long-term goal of our research is to identify those critical elements in these transporters that can make compelling targets for disease intervention. The challenge is to accurately measure interactions between the transporter, which itself changes shape dynamically, and the transported ions or molecules at atomic resolution. We will use computer simulations, which provide atomic-level detail, in combination with a wide range of biophysical, structural, and biochemical experimental techniques to validate and extend the simulation results: For Aim 1: Mechanism of sodium/proton transport in CPA family transporters we will use the prokaryotic model protein NapA and identify sodium ion and proton binding residues and identify triggers of the conformational transition by molecular dynamics (MD) simulations together with thermodynamic measurements of ion binding affinities and the transient currents via electrophysiology together with X-ray crystallography. In order to make results from model protein studies directly available for disease-relevant human transporters we will (Aim 2) create a public database SLC9-DB that integrates structural models of all human CPA transporters in multiple conformations with experimental and simulation data. For Aim 3: Mechanism of bile-acid transport in BASS transporters we determine the precise interaction of the transported bile acid molecule with the prokaryotic ASBTNM transporter during the transport cycle. We will use MD simulations together with a flexible docking approach and validate the findings by binding affinity measurements and structures from X-ray crystallography. Taken together, this research will provide a foundation for rational and targeted intervention in a range of complex diseases that represent a growing health problem.